Negative regulation of oncogenic Ras signaling by mitogen-activated protein kinase phosphatase-3

Activating mutations in the Ras oncogene are found in ∼30% of all human cancers. Ras is a central regulator of intracellular signaling pathways, such as the Raf/MEK1/2/ERK1/2 protein kinase cascade, that modulate cell cycle progression, cell proliferation, and cell survival. Chronic activation of Ras-stimulated pathways is believed to play a key role in cellular transformation, although constitutive activation of Ras alone is not sufficient for tumor development. The goal of these studies is to further understand the biochemical events that occur during carcinogenesis in cells that express activated Ras that make cells susceptible to tumor promoting stimuli. This knowledge is important for the development of new strategies to diagnose, prevent and treat cancer. To date, attempts at developing small molecule inhibitors of oncogenic Ras have been unsuccessful. Therefore, efforts have instead focused on targeting downstream effectors of Ras, such as the Raf/MEK1/2/ERK/12 cascade, or preventing the posttranslational prenylation of Ras that is critical for its function. The aim of these studies is two-fold: (1) to elucidate the signaling events that occur in cells exposed to tumor-promoting stimuli; and (2) to develop tools for the study of prenylation in living cells.;Previous studies in our lab showed that the MAP kinase phosphatase MKP-3 is a vulnerable target of tumor promoting stimuli during carcinogenesis in cells that express activated Ras. Therefore, we sought to further understand the mechanisms that control the expression of MKP-3. We found that MKP-3 RNA and protein expression is intricately controlled by ERK1/2. We also found that MKP-3 is engaged in a negative feedback loop that is important in limiting oncogenic Ras signaling through the Raf/MEK1/2/ERK1/2 pathway. We also previously showed that one tumor promoter known as palytoxin stimulates ERK1/2-dependent changes in gene expression. These studies relied on the use of pharmacological inhibitors of MERK1/2, the ERK1/2 activating kinase. Recent reports have revealed that MEK1/2 inhibitors also inhibit MEK5, the kinase that activates the MAP kinase ERK5. Therefore, we re-evaluated the effect of palytoxin in cells with activated Ras, with a focus on ERK5. We found that palytoxin also triggers the activation of ERK5. Palytoxin-stimulated ERK5 contributes to the upregulation of c-Fos, a signaling event that we previously attributed solely to ERK1/2.;Prenylation of Ras is a post-translational modification that is critical for Ras function. Drugs that disrupt prenylation are therefore promising cancer therapeutics, and several such inhibitors have been tested recently in clinical trials. More information regarding the mechanistic details of protein prenylation and functional consequences of disrupting prenylation in the cell is needed. In a collaborative study with the Distefano lab in the department of chemistry, I tested the uptake and localization of cell-penetrating peptides that will be useful for the study of prenylation in living cells. We found that prenylated peptides are rapidly taken up by cells and localize in a perinuclear fashion. In contrast to previous studies of cell-penetrating peptides, uptake is more dependent on the presence of the hydrophobic prenyl moiety than on the identity of amino acids that make up the peptides. These results, discussed in Chapters IV and V, may be valuable for the development of more specific inhibitors of Ras prenylation.;Altogether, the studies presented in this thesis provide new insights into the biochemical mechanisms involved in the negative regulation of oncogenic signaling pathways. This information may be useful for the development of new ways to treat and prevent cancer.